The goal is to develop new fluorescence markers for automated cytology. Synthesis of poly-aza-xi-A, poly-U-copolymer as fluorescent markers for mRNA and the evaluation by fluorescence microscopy and electron microscopy are now in progress. Combination of DNA and enzyme analysis on the same specimen can also be done by fluorescence-quenching methods recently developed in our laboratory. We have now applied this method to endometrial cancer, which is an ideal model system for comparison of normal proliferation and cancer. One hundred endometrium specimens have been studied with flow cytometry for DNA analysis (FCDA) and 5'-nucleotide phosphodiesterase (5'-NPD), a proliferative enzyme marker. FCDA data showed that aneuploidy was present in only 5 of 40 cancer specimens. With corrected histograms, in the G2/M compartment of all cancer specimens, the percentage of DNA was higher (greater than 5%) than the non-cancer specimens (less than 4%). Thus, FCDA alone can be a useful diagnostic aid for endometrial cancer. The determination of 5'-NPD was done with 5,5'-diiodoindigo as a quencher liberated from the use of 5'-(5-iodo-3-indoxyl) thymidine phosphodiester (IIpT) as an enzyme substrate and 4',6-diamidino-2-phenylindole (DAPI) for DNA. This method could qualitatively define which population of the cell cycle had a higher enzyme level and also quantitatively gave the enzyme units per cell. It was found that 12.5% of all cancer specimens had 5'-NPD activity in the G0/G1 cells and 87.5% in the S and/or G2/M cells, whereas in the non-cancer specimens 5'-NPD was found in 34% of the G0/G1 cells and in 66% of the specimens in the S and/or G2/M cells. In the hyperplasia specimens, 5'-NPD was found in 28.5% of the G0/G1 cells, and 71.5% of the specimens had 5'-NPD in the S and/or G2/M cells. Furthermore, the concentration of 5'-NPD was found to be 5 times higher in the G2/M cells of the cancer specimens than that in the non-cancer specimens. And yet, in the hyperplasia specimens, the activity was found only twice that of the normal specimens. The results of this investigation provided evidence for the first time that this exonuclease activity alters the cell-cycle fractions and that a decrease in the enzyme activity in G0/G1 cells and an increase in G2/M cells may be an indicator of human endometrial cancer. (3)